Since the development of recombinant DNA techniques in the late 1970s, gene fusion technology has been used for generating multifunctional proteins for a broad range of applications. Fusion proteins are utilised in protein science research for diverse applications. In order to construct such fusion proteins, two types of connection are possible. One is ‘end to end’ fusion in which the N-terminus of one domain is linked to the C-terminus of the other domain. The second is “insertional” fusion in which one domain is inserted in-frame into the middle of the other parent domain.
Proteins or fragments thereof are used for active immunization. Active immunization involves trying to stimulate humoral and cellular immune responses by administering antigens. These antigens can be killed or weakened forms of the disease-causing agents, subunits of the infectious agents and recombinant proteins corresponding to the native proteins or to polyepitopes. Passive immunization involves giving antibodies called gamma globulins to someone who was infected with the disease.
A major obstacle in vaccine development is variability, complexity and adaptability of pathogens. For instances, the variability of S. aureus is associated with its twelve serotypes which are related to the capsular polysaccharide composition. The complexity of this microorganism is due to its phenotype because S. aureus is able to produce more that forty virulence factors. The adaptability is related to the capacity of the bacteria to accumulate mutations and to become resistant to antibiotic treatment or even more to escape to the immune system. This explains the tendency of some pharmaceutical groups to develop multivalent vaccine by combining several antigens in a same vaccine formulation. In this context, a vaccine consisting of a single protein would be less costly to manufacture and has the advantage of a practical production process and more straightforward quality control testing than a vaccine consisting of several recombinant proteins.
Epitope characterization is medically important in application such as vaccination and antibodies developments. There are a number of advantages to immunize only with the epitope rather than with the entire organism or with the isolated antigen such as the safety, the difficulties to obtain pure antigens, the problems of immunodominance of some epitopes or the possibility to focus the immune response on protein regions which are of crucial importance for the biological activity of the protein.
If various epitope mapping techniques have permitted to unravel antibody/antigen interactions, the possibilities to deliver these small B cell epitopes to the immune system are lacking. According to the literature, the classical approaches that consist simply to fuse epitopes gives rise to polypeptides that are poorly antigenic. An alternative approach of epitope-vaccines is the conjugation to a carrier able to elicit an efficient T cell help for antibody producing B cells. Consequently, this is this feature that limits drastically the use of epitope-vaccines obtained by gene fusion. In this way, virus-like particles have demonstrated to be suitable molecular scaffold of B cell epitopes. But this approach is often confronted to the “assembly problem” occurring when the epitope fusion or insertion prevent assembling of the core protein to form the virus-like particle.
Staphylococcal alpha-hemolysin (HA) is secreted by most strains of Staphylococcus aureus as a water soluble monomer of 293 residue polypeptide. This toxin forms heptameric pores in membranes of susceptible cells such as platelets, erythrocytes, peripheral blood monocytes, macrophages, keratinocytes, fibroblasts and endothelial cells. HA is endowed with haemolytic, cytotoxic, demonecrotic and lethal properties as well in humans as in animals. A role of this toxin in Staphylococcus aureus biofilm formation and more precisely in cell-to-cell interactions was also reported. The crystal structure of the heptamer HA in detergent indicates a mushroom-shaped object in which the lower half of the stem (K110 to Y148), a 14 strand β-barrel, forms a transmembrane channel made up of residues from the seven central-glycine-rich regions of the polypeptide chains (FIG. 1). The N- and C-terminal third of the polypeptide is organized in a β-sandwich structure that participates to the formation of the cap of the mushroom located outside of the targeted cell.
Staphylococcus aureus is a hardy, Gram-positive bacterium that is frequent cause of both community-acquired and nosocomial disease. Transient asymptomic colonization is common, occurring in up to 40% of children and adults.
In a minority of cases, S. aureus infection is associated with severe disease, including bacteremia, osteomyelitis, endocarditis, meningitis and abscess formation. S. aureus infections are relatively difficult to treat, and invasive diseases and relapse may occur following antibiotic treatment.
The infections caused by S. aureus are also problematic in animals. Bovine mastitis is the most costly disease to the dairy industry worldwide, with losses estimated at 2 billion dollars per year in the United States alone. Mastitis is the primary reason for culling, e.g. mastitis was reported to be the reason for 15% of the cows culled in the USA. S. aureus is one of the most frequent causes of bovine mastitis, and despite several decades of research aimed at controlling this pathogen, it remains a substantial economic problem to milk producers worldwide.
Treatment is made more difficult by the increasing number of strains that are resistant to multiple antibiotics, including methicillin. Resistance to vancomycin, an antibiotic of last resort, has also developed recently. Disease prevention is therefore the best way to limit the morbidity and mortality caused by this organism as well in humans as animals. As whole-cell live or killed vaccines largely fail to generate protective immune responses, alternative immunizations have been tested such as:
Active Immunizations:                Capsular polysaccharides act as virulence factors by reducing the ability of host polymorphonuclear neutrophils to opsonise the bacteria. Although 12 S. aureus capsular polysaccharides have been identified, types 5 and 8 have historically accounted for the majority of S. aureus diseases. A vaccine has been developed that contains type 5 and 8 polysaccharides (StaphVAX®, Nabi Biopharmaceuticals). Its protective capacity has been demonstrated in a mouse model. It has also been shown to be safe and immunogenic in humans. Although it is the only staphylococcal vaccine to date that has been tested in a Phase III clinical trial, it failed to meet the goals of this Phase III clinical trials.        Immunization with poly-N-acetylglucosamine, a S. aureus surface carbohydrate synthesized by icaABC products, has been shown to protect mice against staphylococcal disease.        Subunit vaccines composed of individual surface proteins, for example, clumping factor A (ClfA), clumping factor B (ClfB), iron-regulated surface determinant B (IsdB), or fibronectin-binding protein (FnBP), generate immune responses that afford partial protection against S. aureus challenge of experimental animals.        Passive Immunizations:        
Monoclonal antibodies were currently being tested for their ability to interfere with staphylococcal infections. A formulation containing high levels of opsonising antibodies against S. aureus capsular polysaccharide types 5 and 8 (Altastaph™, Nabi Biopharmaceuticals) is currently in clinical trials.
Teichoic acid is a polymer expressed on the surface of S. aureus that is present in two forms: wallteichoic acid (WTA), a major component of staphylococcal cell wall, and lipoteichoic acid (LTA), which is connected to the cell membrane. A monoclonal anti-LTA antibody is currently being tested for its ability to prevent coagulase-negative staphylococcal sepsis. A phase I/II double-blind, placebo-controlled study of the anti-LTA monoclonal antibody demonstrated that the antibody is safe and tolerable in premature infants and shows linear pharmacokinetics. Efficacy trials have not been reported.
Veronate, a passive immunotherapy developed by Inhibitex, based on the humanized monoclonal antibodies recognizing the S. aureus ClfA protein and S. epidermidis SdrG. These monoclonal antibodies show some protective efficacy in animal models of S. aureus infection.
The document WO2007/145689 A1 refers to vaccines comprising an S. aureus alpha-toxin and a pharmaceutically acceptable carrier, wherein the S. aureus alpha-toxin antigen may contain at least two alterations that reduce its toxicity and/or may be conjugated to or co-administrated with another bacterial antigen. The vaccines may comprise one or more other bacterial antigens. According to WO2007/145689 alpha-hemolysin protein alone is used as antigen and/or is chemically conjugated with other S. aureus antigens. However, the disadvantage of conjugated proteins is that cross-linking will compromise structure, activity, immunogenicity and antigenicity. Further, each protein component to be mixed together or to be conjugated has a different purification history due to different physico-chemical properties, has a different degree of purity, and possibly has different impurities In addition, it is difficult to control the stoechiometry of the chemical conjugation between the different proteins leading to a problem of heterogeneity in the protein solution. Therefore, maintaining the same quality of different charges is difficult. In addition, conjugation of proteins result in a mixture of different products which can not properly defined.